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1. Introduction

Context

Siem Reap, Takeo, Battambang and Kampong Cham clinical microbiology laboratories based at Provincial Referral Hospitals (250-300 beds), receive technical and supply support such as long term mentoring, culture media and diagnostic reagents from the Diagnostic Microbiology Development Program (DMDP).

Culture media including blood culture bottles, agar plates and biochemical tubed media are produced centrally at the Central Media Making Laboratory (CMML) at the University of Health Sciences, Phnom Penh. CMML, ISO 9001:2015 certified, performs media quality control before distribution to the clinical laboratories.

Laboratory procedures

a. Specimen processing

Clinical laboratories use a manual blood culture system (CMML). Blood culture bottles are registered and staff measure blood culture volume by weighing the bottles. Air venting needles are inserted before incubation for 7 days. After overnight incubation at 35oC, laboratory staff inspect bottles daily for signs of organism growth such as turbidity, bubbles or hemolysis. Staff perform blind sub culture and Gram stain at 24h and terminal subculture (or Gram stain) at day 7. If positive, laboratory staff notify clinician immediately.

Fig 1. Manual blood culture bottle processing

b. Bacterial identification

If organisms are detected, microbiologists will perform further identification by using Standard Operating Procedures and Job aids. Example, Dr. Ellen Jo Baron and DMDP flowcharts for identification of Burkholderia pseudomallei and Staphylococci.

Fig 2. Identification of Burkholderia pseudomallei

c. Antibiotic Susceptibility Testing

Antibiotic Susceptibility Testing follows Clinical and Laboratory Standards Institute (CLSI) performance standards, M100 and M02 for disk diffusion testing. MIC using gradient diffusion (Biomerieux) is used for selected antibiotic organism combinations.

Fig 3. Antibiotic Susceptibility Testing and interpretation

Data management

Patient information and laboratory data are registered in the Cambodian laboratory information system (CamLIS). Every month microbiologists analyse data and communicate findings with local hospital healthcare professionals. Raw data are extracted from CamLIS as an Excel file and stored in a folder “Microbiology Report/data”. Data analysis was conducted by using R Markdown with pre-writing plain texts and R programming language version R 4.1.2. Packages tidyverse 1.3.1, AMR 1.8.0, janitor 2.1.0, readxl 1.3.1, plotly 4.10.0, kableExtra 1.3.4 were used. The report from R Markdown is generated in HTML format which can be opened with any web browser in smartphone or computer. e.g Google Chrome, Safari etc.

2. Patient demographic

We assume a patient receives care at a hospital (inpatients and outpatients) or private clinics and has only one patient identification number. We removed duplicates to find the actual number of patient requests. Thus, we have: 1,223 patient(s).

2.1. Gender

The proportion of patients by gender

2.2. Age

Distribution of age group and gender. We accepted the range 0 to 110 years old. We have 1,218 patient(s)

3. Microbiology specimen

A patient can have more than one microbiology specimen collected. We assume one specimen has one unique sample identification number. After removing duplicates, we have: 1,644 sample(s). We stratify data by month and sample type

For further insight, we stratified by sample sources

Specimen rejection

Specimen Total specimen Total reject Reasons
Blood Culture 759 1 No sample
1 ប័ណ្ណស្នើសុំវត្ថុវិភាគមាន២
Pus 169 1 No label
Sputum 49 16 > 10 epithelial cell/LPF
1 No sample
Urine 350 2 No sample

4. Cerebrospinal fluid (CSF) pathogens isolated

There were 27 CSF positive.

5. Blood culture

Blood culture is defined as a critical specimen. Data from blood culture can be used for quality improvement and developing guidelines, surveillance of emerging pathogens and antibiotic resistance. Only the first of each pathogen for each patient in the period of 30 days was counted. Patient identification, collection date and organism name are used to find the first isolate.

5.1. Blood culture request by patient and true pathogen rate

Patient identification and blood samples were filtered to define the number of patient requests by month. We have 704 patient(s) blood culture requests. Calculate true pathogen rate:

formula calculate true pathogen

The true pathogen rate expected range is 6% to 12%.

5.2. Bloodstream pathogens isolated

Pathogen Jan Feb Mar Apr Total
Escherichia coli 6 3 6 6 21
Burkholderia pseudomallei 4 2 7 4 17
Acinetobacter 1 2 5 2 10
Cryptococcus 5 2 2 1 10
Klebsiella pneumoniae 2 2 1 0 5
Staphylococcus aureus 1 2 2 0 5
Burkholderia cepacia 1 0 0 0 1
Elizabethkingia 1 0 0 0 1
Moraxella 1 0 0 0 1
Pseudomonas aeruginosa 1 0 0 0 1
Stenotrophomonas maltophilia 1 0 0 0 1
Streptococcus pneumoniae 1 0 0 0 1
Streptococcus suis 1 0 0 0 1
Listeria monocytogenes 0 1 0 0 1
Ochrobactrum 0 1 0 0 1
Enterococcus 0 0 1 0 1
Pantoea 0 0 1 0 1
Proteus mirabilis 0 0 1 0 1
Salmonella Typhi 0 0 1 0 1
Streptococcus pyogenes 0 0 1 0 1
Streptococcus, beta-haem. Group B 0 0 1 0 1
Non-fermenting gram negative rods 0 0 0 1 1
Salmonella 0 0 0 1 1
Shigella sonnei 0 0 0 1 1
Streptococcus anginosus group 0 0 0 1 1

5.3. Blood culture bottle collected and contamination rate

Blood culture contamination can lead to inappropriate treatment of patients, over use of resources and a workload burden. Microbiologists are obliged to monitor and report contamination rate regularly to specimen collectors. We defined contaminant as Coagulase negative Staphylococci, Corynebacterium sp., Streptococcus viridans, alpha-hem., Micrococcus sp..

Contamination_formula

The expected contamination rate is < 3%.

NB: To count total number of blood culture bottle collected, we use the blood culture volume field. If blood culture volume is not recorded in CamLIS, the total number of blood culture collected will be lower and the contamination rate will be higher then the real situation.

Organism Jan Feb Mar Apr Total
Coagulase Negative Staphylococcus 9 7 11 9 36
Bacillus 0 3 2 5 10
Corynebacterium 0 1 0 1 2
Micrococcus 0 0 0 0 0
Streptococcus viridans, alpha-hem. 0 0 0 0 0

5.4. Blood culture true pathogen rate and contamination rate by sample sources

Sample Source Patient request True pathogens (%) Number of bottle Contamination (%)
SP 193 36 (19 %) 387 24 (6 %)
Med A 93 10 (11 %) 216 7 (3 %)
Med C 72 5 (7 %) 150 3 (2 %)
Angkor Sante clinic* 71 7 (10 %) 130 2 (2 %)
Royal Angkor * 64 3 (5 %) 74 4 (5 %)
Neonatal 46 4 (9 %) 45 0 (0 %)
ICU Med 44 6 (14 %) 104 5 (5 %)
Med B 44 3 (7 %) 96 1 (1 %)
ICU Chir 34 9 (26 %) 82 1 (1 %)
Chi A 10 1 (10 %) 22 0 (0 %)
Chi C 9 0 (0 %) 20 0 (0 %)
Chi B 5 0 (0 %) 10 0 (0 %)
Psakrom clinic* 5 0 (0 %) 5 0 (0 %)
Kong Rithy clinic* 4 1 (25 %) 8 0 (0 %)
OPD 3 0 (0 %) 5 0 (0 %)
Biomed lab * 2 0 (0 %) 4 0 (0 %)
MCMC 2 1 (50 %) 3 1 (33 %)
Med C Pneumon II 1 1 (100 %) 1 0 (0 %)
Pediatric 1 0 (0 %) 1 0 (0 %)
គ្លីនិក ១០២ 1 0 (0 %) 2 0 (0 %)
* Specimen from private clinc/laboratory

5.5. Blood culture volume

Blood culture volume is vital to detect organism growth. For adult patients, two bottles are collected from two different venipunctures with the target of 8-12ml of blood and pediatric patients, one bottle collected with the target of 1-6ml of blood. One patient may grow two organisms and is represented by two rows in the CamLIS database. One sample has only one unique laboratory identification number. Therefore, we removed blood culture volume duplicates by sample laboratory identification number.

5.5.1. Adult

5.5.2. Pediatric

5.5.3. Adult and Pediatric

4 bottles empty blood culture volume in CamLIS and it is impacted on the calculation of contamination rate

Missing blood culture volume
Month Bottle A Bottle B Total
Jan 0 0 0
Feb 1 1 2
Mar 2 0 2
Apr 0 0 0

5.6. Blood culture day of positivity

Microbiology laboratories always report the day of blood culture positivity in the patient report comment. We present the common pathogens isolated. 1 patient had no record of the day of positivity, therefore, it was excluded in the analysis.

5.7. Blood culture TurnAround Time

TurnAround Time (TAT) is the expectation time that laboratory report result back to collectors. According to Excel extracted from CamLIS, we can report positive blood culture TAT from specimen collection to Notify Gram stain.

2 records missed document date or time and excluded in the analysis.

5.8. Blood culture critical result reporting

Positive blood cultures are life threatening for patients and require immediate reporting.
We analysed data and found 100% of 135 critical results were recorded in CamLIS as reported.

6. Notifiable and other important pathogens list

Pathogens per patient from all specimen types

Pathogen Jan Feb Mar Apr Total
Burkholderia pseudomallei 8 6 8 5 27
Streptococcus suis 1 0 0 2 3
Listeria monocytogenes 0 1 0 0 1
Salmonella Typhi 0 0 1 0 1
Salmonella 0 0 0 1 1
Salmonella Paratyphi A 0 0 0 0 0
Vibrio cholerae 0 0 0 0 0
Bacillus anthracis 0 0 0 0 0
Yersinia pestis 0 0 0 0 0
Francisella tularensis 0 0 0 0 0
Corynebacterium diphtheriae 0 0 0 0 0
Neisseria gonorrhoeae 0 0 0 0 0
Neisseria meningitidis 0 0 0 0 0
NB: Salmonella were confirmed by Salmonella O Polyvalent Antisera serology

Screening for multi-drug resistant organisms isolated from all specimen types
- Cefoxitin resistance for methicillin-resistant staphylococcus aureus (MRSA)
- Vancomycin for vancomycin intermediate staphylococcus aureus (VISA) or vancomycin resistance staphylococcus aureus (VRSA)
- Ceftriaxone and/or ceftazidime non susceptible for Extended Spectrum Beta-lactamase (ESBL) Escherichia coli and Klebsiella pneumoniae. A small number of isolates will be missed using this criteria.
- Imipenem and/or meropenem non susceptible for Carbapenem-resistant (CRE) Escherichia coli and Klebsiella pneumoniae

Organism Mechanisms Jan Feb Mar Apr Total
Escherichia coli ESBL 11 5 14 14 44
Staphylococcus aureus MRSA 5 1 1 4 11
Klebsiella pneumoniae ESBL 3 2 1 3 9
Escherichia coli CRE 0 0 1 2 3
Klebsiella pneumoniae CRE 1 0 0 0 1

Tracking blood culture isolates by month for possible Healthcare Associated Infection (HAI). We filtered blood culture collection date after 48 hours of admission with common bacteria associated with HAI.

3 records excluded due to no admission or collection date.

Organism Jan Feb Mar Apr Total
Klebsiella pneumoniae 1 1 0 0 0
Pseudomonas aeruginosa 1 0 0 0 0
Burkholderia cepacia 1 0 0 0 0
Acinetobacter 0 2 3 2 0
Stenotrophomonas maltophilia 0 0 0 0 0
Pseudomonas 0 0 0 0 0
Non-fermenting gram negative rods 0 0 0 0 0
Staphylococcus aureus 0 0 0 0 0

7. Burkholderia pseudomallei (Bps)

There were 28 Burkholderia pseudomallei reported.

Antibiotic S (%)[95CI] I (%)[95CI] R (%)[95CI] Total
Amoxicillin/clavulanic acid 28 (100%)[90-100] 0 (0%)[0-10] 0 (0%)[0-10] 28
Ceftazidime 28 (100%)[90-100] 0 (0%)[0-10] 0 (0%)[0-10] 28
Meropenem 28 (100%)[90-100] 0 (0%)[0-10] 0 (0%)[0-10] 28
Trimethoprim 28 (100%)[90-100] 0 (0%)[0-10] 0 (0%)[0-10] 28
Gentamicin 0 (0%)[0-10] 0 (0%)[0-10] 28 (100%)[90-100] 28
S: susceptible, I: intermediate, R: resistant

8. Salmonella

There were 2 Salmonella species reported.

9. Cumulative Antibiotic Susceptibility Testing (cAST)

We filter blood culture by first pathogen isolated in an period of 30 days and apply all EUCAST expert rules for Intrinsic Resistance and Unusual Phenotypes (AMR v3.3, 2021) before analyzing antibiotic susceptibility patterns. We produced cAST for pathogens included in the GLASS priority pathogen list.

9.1. Staphylococcus aureus

There were 5 Staphylococcus aureus reported.
Antibiotic S (%)[95CI] I (%)[95CI] R (%)[95CI] Total
Cloxacillin 5 (100%)[60-100] 0 (0%)[0-40] 0 (0%)[0-40] 5
Cefazolin 5 (100%)[60-100] 0 (0%)[0-40] 0 (0%)[0-40] 5
Erythromycin 1 (20%)[0-60] 1 (20%)[0-60] 3 (60%)[20-90] 5
Clindamycin 2 (40%)[10-80] 0 (0%)[0-40] 3 (60%)[20-90] 5
Tetracycline 4 (80%)[40-100] 0 (0%)[0-40] 1 (20%)[0-60] 5
Chloramphenicol 4 (80%)[40-100] 0 (0%)[0-40] 1 (20%)[0-60] 5
Trimethoprim 5 (100%)[60-100] 0 (0%)[0-40] 0 (0%)[0-40] 5
S: susceptible, I: intermediate, R: resistant

9.2. Escherichia coli

There were 21 Escherichia coli reported.
Antibiotic S (%)[95CI] I (%)[95CI] R (%)[95CI] Total
Ampicillin 2 (10%)[0-30] 1 (0%)[0-20] 18 (90%)[70-100] 21
Amoxicillin/clavulanic acid 13 (60%)[40-80] 2 (10%)[0-30] 6 (30%)[10-50] 21
Ceftriaxone 6 (30%)[10-50] 0 (0%)[0-20] 15 (70%)[50-90] 21
Ceftazidime 11 (50%)[30-70] 3 (10%)[0-30] 7 (30%)[20-50] 21
Meropenem 20 (100%)[80-100] 0 (0%)[0-20] 1 (0%)[0-20] 21
Imipenem 19 (90%)[80-100] 0 (0%)[0-20] 1 (0%)[0-20] 20
Gentamicin 13 (70%)[40-80] 0 (0%)[0-20] 7 (30%)[20-60] 20
Amikacin 21 (100%)[80-100] 0 (0%)[0-20] 0 (0%)[0-20] 21
Ciprofloxacin 3 (10%)[0-30] 1 (0%)[0-20] 17 (80%)[60-90] 21
Trimethoprim 2 (10%)[0-30] 2 (10%)[0-30] 16 (80%)[60-90] 20
Chloramphenicol 21 (100%)[80-100] 0 (0%)[0-20] 0 (0%)[0-20] 21
S: susceptible, I: intermediate, R: resistant

9.3. Klebsiella pneumoniae

There were 5 Klebsiella pneumoniae reported.

Antibiotic S (%)[95CI] I (%)[95CI] R (%)[95CI] Total
Ampicillin 0 (0%)[0-40] 0 (0%)[0-40] 5 (100%)[60-100] 5
Amoxicillin/clavulanic acid 3 (60%)[20-90] 0 (0%)[0-40] 2 (40%)[10-80] 5
Ceftriaxone 1 (20%)[0-60] 0 (0%)[0-40] 4 (80%)[40-100] 5
Ceftazidime 1 (20%)[0-60] 1 (20%)[0-60] 3 (60%)[20-90] 5
Meropenem 4 (80%)[40-100] 0 (0%)[0-40] 1 (20%)[0-60] 5
Imipenem 4 (80%)[40-100] 0 (0%)[0-40] 1 (20%)[0-60] 5
Gentamicin 4 (80%)[40-100] 0 (0%)[0-40] 1 (20%)[0-60] 5
Amikacin 5 (100%)[60-100] 0 (0%)[0-40] 0 (0%)[0-40] 5
Ciprofloxacin 0 (0%)[0-40] 0 (0%)[0-40] 5 (100%)[60-100] 5
Trimethoprim 1 (20%)[0-60] 1 (20%)[0-60] 3 (60%)[20-90] 5
Chloramphenicol 3 (60%)[20-90] 0 (0%)[0-40] 2 (40%)[10-80] 5
S: susceptible, I: intermediate, R: resistant

9.4. Pseudomonas aeruginosa

There were 1 Pseudomonas aeruginosa reported.

Antibiotic S (%)[95CI] I (%)[95CI] R (%)[95CI] Total
Ciprofloxacin 1 (100%)[10-100] 0 (0%)[0-90] 0 (0%)[0-90] 1
Ceftazidime 1 (100%)[10-100] 0 (0%)[0-90] 0 (0%)[0-90] 1
Meropenem 1 (100%)[10-100] 0 (0%)[0-90] 0 (0%)[0-90] 1
Gentamicin 1 (100%)[10-100] 0 (0%)[0-90] 0 (0%)[0-90] 1
Amikacin 1 (100%)[10-100] 0 (0%)[0-90] 0 (0%)[0-90] 1
S: susceptible, I: intermediate, R: resistant

9.5. Acinetobacter

There were 10 Acinetobacter reported.
Antibiotic S (%)[95CI] I (%)[95CI] R (%)[95CI] Total
Ciprofloxacin 6 (60%)[30-80] 1 (10%)[0-40] 3 (30%)[10-60] 10
Ceftazidime 6 (60%)[30-80] 0 (0%)[0-30] 4 (40%)[20-70] 10
Meropenem 10 (100%)[70-100] 0 (0%)[0-30] 0 (0%)[0-30] 10
Gentamicin 5 (50%)[20-80] 1 (10%)[0-40] 4 (40%)[20-70] 10
Amikacin 9 (90%)[60-100] 0 (0%)[0-30] 1 (10%)[0-40] 10
Trimethoprim 5 (50%)[20-80] 0 (0%)[0-30] 5 (50%)[20-80] 10
S: susceptible, I: intermediate, R: resistant

10. Acknowledgement

We gratefully acknowledge support from Siem Reap Provincial Referral Hospital, Bureau of Medical Laboratory Services (BMLS), Ministry of Health Cambodia, Laboratory Strengthening Partners, the Defense Threat Reduction Agency (DTRA) and the Diagnostic Microbiology Development Program (DMDP).